FSI Flashcards
octamer of histones + spiral/solenoid stability
h2a, h2b, h3 and h4
h1 binds nucleosome and linker DNA. may stabilize spiral/solenoid stuff through interaction with histone N terminal tails
where does HAT acetylate and how does it work
transfer acetyl to NH3+ group of lysine
removes the + charge on histones, decreasing strong interaction with - PO4 dna groups
“writer”
general transcription factors
locate tata, define start of transcription, recruit rna pol 2, low rate of transcription with no specificity, present in all cell nuclei
proximal promoter, consensus sequence
tata box
- meaning that it is found in a lot of things
- close to a promoter region
- binds specific transcription factors (tissue spec)
- approx 18-26 bases upstream
timing of transcription initiation specific factors
chromatin remodelling, histone mod, TBP finds tata, then TF2D, then B, E, H (general transcription factors that guide pol 2). guide the formation of the basal transcription complex
regulatory transcription factors
control rate of transcription, function as activators or repressors, often tissue specific
transcription process
tata box recognition, activation/dna binding/dimerization, a helix binds to groove bending dna and separating strands, basic/hydrophobic AA (leu) stabilize DNA binding
zinc finger motif transcription factor
zinc finger binds to specific motifs like 2 cys/2 his
- activation by ligands like hormones binding to receptor
- binds in major groove and forms a stretch of a helices
- zinc holds a helix and b sheet together. cluster of 3 that form in major groove and have a helix to connect. strong and specific
When it needs to open the DNA sequence, it needs tighter binding so you need more than one protein to accomplish this
dna binding domain in transcription factor
recognizes consensus dna sequence
-a helix with basic aa, lies in major or minor groove and contacts n bases (DIRECTLY), basic aa stabilize binding
- bend dna!
activation domain in transcription factor
interacts with other tx factors
domains of transcription factors
activation, dna binding, dimerization (from N to C terminus)
bzip
DNA binding: each subunit has 1 a helix. a helices with basic aa that contact n bases in major groove. basic aa stabilize them
dimerization: amphipathic a helix. dimerizes in coiled coil by hydrophobic interactions
ex. leucine zipper
leucine zipper dimerization domain
every 7th aa is leucine. heptad repeat
provides amphipathic a helix for dimerization
- other hydrophobic aa could replace leu
ap1 transcription factor
activator protein 1
- bzip protein!
- ubiquitous tx factor
- regulates gene expression in response to growth factors, stress, pathogens
- controls cell processes including differentiation and cell division
- activation domain interacts with other tfs
nuclear receptor family dimer proteins
- must be acticated by ligand binding (lipid sol hormones and vitamins)
- coordinate the response of many genes to a hormone signal
- enter by active diffusion and bind to intracell receptors
- receptors are conserved!
dna binding domain of GR
nuclear receptor family
- each subunit has 2 zinc fingers
- 1 a helix of 1 zinc finger lies in major grooce and binds to similar sequence in dna. thus its 2 half sites
- there’s a lever arm that moves
can activate many genes at once as long as they have the GRE
myogenesis
done by myoD, myf5 and myogenin
- HLH protein
- binds reg region in many muscle specific genes, conserved
bone formation
runx2
- binds to enhancers
- runt domain clamps the dna between c terminal tail in major groove and the wing in minor groove
- mutation is cleidocranial dysplasia
MRNA cap
xtra nuc added to 5’ end. 7 methyl guanosine. added by 5’ to 5’ tiphosphate bond
- added as primary transcript emerges (not encoded)
- increases stability and required for translation
- polio target, makes itself without CBP!
intron removal and ligation esterification
2 transesterification reactions
- 2’ OH of one site attacks the 5’ splice site
- 2’ OH is attached to 5’ site
3’ OH upstream attacks 3’ splice site
exons joined by ligation
intron removal, protein side
large RNP with small nuclear RNP that have specific small nuclear RNA that are complementary to consensus sequences in precursor rna
- help in poitioning the spliceosome on precursor rna
binding
- at 5’ end and branch point. then form spliceosome then 5’ site is cleaved, 3’ is cleaved
poly A tail
protien binds AAUAAA
- cleavage factors CFI and CFII cleave rna
- poly A polymerase adds poly A posttranscriptionally
- increases stability and translational efficiency
miRNA processing
transcribed by pol 2 or intron derived
- regulate post transcriptionally
- each mrna has many target sequences for miRNA
- first forms pri-mirna from pol 2 or intron by forming hairpin structure
- processed by drosha in nuc forming pre-miRNA
- dicer cuts it in cyto
one strand is selected, other is degraded. assemble them into microRNPS or miRISC (from argonaute family)
- reg dna expression by destabilizing mrna or interfering with translation
moving from transcription to translation in cyto
cap binding protein.
mRNA then associates with cap and poly a binding proteins to form mRNPs (aka exon junction complex). mRNPS are transported thru nuclear pore from 5’ end then the cap is removed and eIF4 initiation factor is added
5’ non coding region of mrna
determines translational efficiency
protein coding sequence mrna
aka open reading frame. starts at aug and stops at stop codon
3’ noncoding mrna
influences rna stability
ribozyme
28 s that catalyzes peptide bond formation
translation factors
to load aa on tRNA requires ATP, 2 gtp for binding tnra to ribosome and translocation
eIF is initation: bind iniation tRNA, bind mRNA cap and associate mRNA with 40s and unwind mRNA. need GTP
- eEF is elongation that delivers trna to a site, translocate ribosome
- termination factors make stuff fall off
translation binding steps
- eIF2 binds initiator tRNA
- initiation tRNA binds to small ribosomal subunit
- cap binding complex, eIF-4E, G binds mRNA
- complex of 40S subunit tRNA binds to 5’ end of mRNA
- complex scans mRNA until initiator tRNA finds first aug
- anticodon of initiator tRNA is complementary to AUG
- large ribosomal subunit joins to form 80s complex
- initator trna is in p site
stops by release factors
streptomycinn
changes shape of 30s trna so mrna is read wrong
tetracycline
stops trna anticodon reading of mrna codon
erythromycin
binds to 50 s rRna and prevents elongation
chloramphenicol
stops 50S rrna and inhibits peptide bond formation
secretory pathway
proteins that are synthesized on ribosomes associated with ER. include extracell matrix, growth factors/hormones, cell surface receptors
- KDEL
- need to retain stuff here since post trans mod happens
mitochondria signal sequence
the matrix is the longest one
- usually hydrophobic amphiphillic alpha helix binds to hydrophobic face of the receptor
- use tom and tim to get inside
ubiquitination
added on C terminus to lysine by e 1,2,3 and ligase. many ubiquitins added
- proteasome degrades them by using cap enzyme to bind, atp hydrolysis to unfold proteins and feed them into a cylinder that is digested by proteolytic enzymes
**cytoplasmic pathway! meaning if there are misfolded proteins in ER, they are translated back through the translocon for degradation in cyto
ubiquitination signals
hydrophobic region on protein sufface is a sign is misfolded
- cyclins have odd sequence after they are done their job
phosphorylation by kinase causes conformational change exposing recognition sequence
common lymphoid progenitor cell makes
b and t cell
bone marrow to lymph node
T mature in thymus, active in lymph, roam in blood and lymph
B mature in bone marrow
common myeloid progenitor cell makes
neb, mast cell, monocyte
bone marrow to blood then tissue
cytotoxic t cell
bind to MHC class 1 on infected cell
- cd8
- self destroy after binding pathogen by lysis or apoptosis
helper t cell
bind to MHC class 2 on APC
- cd4
- release cytokines to activate b and cytotoxic t cells
- mature in thymus
antigen presenting cells
bone marrow derived: dendritic cells (phago and pinocytosis, fly to lymph for apc, make cytokines), langerhans, macrophages, b cells
have both mhc 1 and 2!
b cell stays in lymph
**follicular dendritic cell found in lymph node
have costim markers such as cd80 (b7-1_and cd 86 (b7- 2)
- recognition phase
adaptive and innate both do it. involves dendritic cells that recognize pathogen, process antigen and present. nonspecific inflam
- effector or response phase
via cytokines, neutrophils, macrophages (innate), antibodies and effector t lymph (adaptive) eliminate antigen
proinflam cytokines
innate or adaptive, mostly macrophages
- involve IL 1,2,6
TNF 1, INF a, INFb
- upregulate inflam reactions
regulatory cytokines
immunosuppressive: IL 10 and TGF b reduce expression of costim receptors that inhibit immune cell growh and stop response using t cell stim then t reg stim
chemokines
chemotaxis. recruit phagocytes to site of inflam like neutrophil, macrophage, fibroblast using IL 8
adaptive immune
key is lymph and antibodies. not actie unless antigenic challenge with high specificity
b cell
- after apc up, t helper cell binds, secretes cytokines, activates b cell then does clonal expansion and antibody production
T helper and APC
- acp binds to t helper (MHC 2) OR t cytotoxic binds to MHC 1
antibody structure
binds antigen at specific recog site and its constant regions activate complement through the classical pathway
- variable regions at the ends of the Y
- 2 heavy chain and 2 short light chains held by disulfide bonds
IgG
smallest, most abundant, mostly in tissue but in blood too
IgM
first to be made by b cells in infection, pnetamer so biggest
igA
in secretions, forms dimers
igE
minute amounts, primarily involved in allergy. constant region interacts with basal and mast cells
igD
stays on B cell (receptor thing)
least found
ways that antibodies stop virus
- neutralize = physical block of pathogenic parts
- agglutination = clump by blocking epitopes
- tag for destruction = signal the complement, phago, NK
- precipitation = makes them insoluble
- inflam = triggers histamine so more immune cells move
- complement = pokes holes in cell by binding to antibody, cascade (classical pathway)
*opsonization is enhancing phagocytosis!
cystic fibrosis, sickle cell, phenylketonuria, congenital deafness and recessive blindness
autosomal revessive
3 ways of getting autosomal dominant disorder
hapoloinsufficiency (missing gene so make less), dominant negative effect (make a toxic protein that also blocks health protein), gain of function (make too much protein)
familial hypercholesterolemia
haplo
- insufficient product made for ldlr resulting in high cholesterol or even very high in homozygotes (heart attacks in childhood)
osteogenesis imperfecta
dominant negative. more than 50% decrease in protein activity because its blocked by toxin
achondroplasia
autosomal dom with gain of function
- fibroblast growth factor receptor 3 (FGFR3)
- membrane bound receptor for fibroblast in chondrocytes and osteoblasts to regulate bone growth
- it is negative inhibition of growth by stopping the prolif of chondrocytes that produce cart
- this means - effect on endochondral ossification
hemophilia
xlinked recessive
x linked dominant on pedigree
no male to male transition
- id between autosomal dominant inheritance
amelogenesis imperfecta gene defects
mutations in enamelin gene (ENAM) = autosomal dominant
- MMP20 = autosomal recessive
- amelogenin gene (AMELX) = x linked pattern where men are more severe than women
amelogenesis imperfecta symptoms
thin enamel, yellow teeth and weak, smaller and cracked teeth, tooth decay/loss, open bite malocclusion
chromosomal disorders
change in number or structure
- down syndrome is trisomy 21
- turner is monosomy of x. always female
complex inheritance
polygenic. environmental factors and many genes play a role
- tested using familial clustering (family vs population average) and rate of occurance between twins and full siblings
- cleft lip and palate
cleft lip and palate
- incomplete fusion of lip and palate
- asians, boys, unilateral is common
- genetics: number of relatives with it, wonky chromosomes, IRF6 mutations (normal formation of lips, palate skin)
- environmental: drugs, meds, nutrition, infection during preg or illness
polymorphism
- two or more varient forms of trait on one gene
- mutation in coding is rare and harmful, but noncoding is not hamful
- usually point mutations
- periodontal disease
periodontal disease
- polymorphism
- we all respond diff to meds because of microbiome, genetic and epigenetic, eco etc
- has to do with IL1 and IL6 that do inflam
- no testing other than probing depth and 2q1
hypodontia, supernumerary teeth, malocclusion, hereditary gingival fibromatosis, oral cancer
- missing 1-5 teeth
- usually with other disease like cleft lip, cleidocranial dysplasia (bone and tooth disease), garnder syndrome
- overbite is overlap btw upper and lower incisors, overjet is max first
- overproduction of collagen
- inherited mutations carry higher risk of cancer. fanconi anemia is rare recessive disorder that can increase risk
type 2 dia, sickle cell anemia, osteogenesis imperfecta, epidermolysis bullosa, amelogenesis imperfecta
- increase risk of perio disease
- vulnerable to infection, problems with anesthesia
- alterning bone and dentin
- blistering of mucosa, making oral health bad and hard for dentures
- enamel structure off
haplotype
set of alleles at two or more neighboring loci on one of the two homologous chromosomes
compound heterozygote
two different mutant allells of a gene, not one wild type and one mutant
mendel’s law
3:1 ratio
segretation is that one of two gene copies are distributed to each gamete
- law of independent assortment: games get genes independently from other gene sorting
- mitosis gets diploid chromosomes, meiosis gets haploid (homologous chromosomes separate)
- chromosome theory of inheritance: traits are controlled by genes residing on chromosomes transmitted thru gametes
humoral
in lymph, happens it path bypasses other apc
- mhc 2
- b cells make free floating antibodies after stim from helper t
cell mediated
t cell (requires 3 signals for activation 1) through antigen 2) through CD28)
binds apc then release cytokines, activate t helper (transforms b cell to plasma cell) and makes cytotoxic t
t cell signalling steps and receptors on t and b cells
first signal comes through antigen receptor, second comes through CD28, last is through cytokines
- apc with mhc 2 turns on the cd28 costim receptor on t helper
- b cell turns it on via mhc 2 but also costim receptor 40
1st signal of t cell
T cell receptor (TCR) has xcell a and b chains that interact with APC
- also has coreceptors cd3
- all of these intracell domains have ITAMS (immunoreceptor tyrosine based activation motifs)
- itam is kinase that needs p to be on
- binding of CD4 or CD8 to MHC complex since they have lck kinase that adds p to ITAM on TCR and CD3
- then ZAP 70 molecules are phosphorylated by lck
- this activates 4 primary signalling molecules of t cell
what does ZAP-70 activate in t cell
first phosphorylates LAT and SLP - 76
- it turns on PI3k that makes PIP2 –> pip 3 (a 3rd kinase)
- this first recruits kinase akt to plasma that influences cell meta to accommodate new energy demand of active t cell
- 2nd, turn on PLC gamma enzymes that allow translation of cytokines and stuff
- 3rd is activation of vav that rearranges cytoskeleton
- 4th is recruiting adap that enhances t cell trafficking
2nd signal of t cell
co stim markers such as CD80 on APC can bind go CD28 on T cell
- CD 28 of t helper can bind CD80 (b7-1_and cd 86 (b7- 2)
- CD 27 (diff receptor than apc)
- 4-1BB has its ligand on apc from t helper. this triggers t cell activation and expansion of cytotoxic t
- bb also increase tH1 cytokines and suppress th2 cytokines
how are antibodies made when antigen present
- carb antigen
Toll like receptor that then makes muliunit antibodies (only a few like M and G2)
- need t cell to do class switch and make diff antibodies
how are antibodies made when antigen present
- protein antigen
b cell recognises path. MHC 2
- 1 helper cell binds with MHC, binds CD40 then cytokines bind, class switch to make all the antibodies
- also prolif and diff into plasma cells
vaccines
- have adjuvant
- APC binds and presents antigen of vaccine, activate cd4 and 8, recruit b cell and prolif or lyse cell
- b cell also then forms long lived plasma cell in bone marrow!
adjuvant
- have adjuvant that essentially protects the antigen and keep it localized
- the adjuvant and antigen are both endocytosed
- also activates toll like receptor that increases transcription factors, cytokines to activate Th1 and 2
- activates inflammasome that increase immune response via ALUM?
- activates Th1 for cell mediated immune response (opsonizing antibodies that tell immune where path is)
- activates TH2 that do humoral response, make neutralizing antibodies that surround and block pathogen!
**alum is hydrophobic and makes a bubbnle
th1 and th2
- Th1 for cell mediated immune response (opsonizing antibodies that tell immune where path is)
- TH2 that do humoral response, make neutralizing antibodies that surround and block pathogen!
crisper
crispr is dna snip and cas 9 is enzyme, essentially guide rna tells where to bind to target sequence. its a replica of the genome to edit
- cas 9 follows and makes a cut on BOTH strands of dna
- dna pol recognizes the damage and tries to repair it
dna methylation
occurs in cytosine-guanine (CpG) region
- methyl = no transcription (silencing) “writer”
- happens after replication
- always on c5 of cytosine!
- frequenly paired with deacetylated histones
- altered gene means cancer, with hypometh in genomic segmnets and hypermeth in others
- in promoter region!!!!!!
**molecular basis of imprinting where you need differentially methylated region. usually only one allele is methylated. the other is expressed!
histone varients
different histones for diff functions, that replace some parts of core histones to make special chromatin structures or mark regions
- ex. H2a.X is varient in response to dna damage that require repair
primary dmr
germline. acquire methylation during gametogenesis
- this is like female imprint rewritten in paternal pattern in sperm or mom genes in egg when it comes to the germ cell
secondary dmr
acquire methylation post fert in differentiation of cells
- ex you add DNMT3a and 3b a methyl group to get differentiation
- or use dna methyltransferase (DNMT) to silence pancreas dna in liver cells
- maintained in cell division by DNMT1
epigenetics and cancer
in normal cells, promoter dna meth is associated with silencing, in cancer cell, meth pattern changes so there is hypo and hyper
- hyper in promoter region can lead to tumor suppressor gene inactication!! that means its silenced and alters expression
- drug vidaza for leukemia
epigenetics in dentistry
ortho: craniofacial deelopment
- use diagnostic kits to target growth problems. occlusion is not always from teeth but other factors
- oral malformations like cleft lip and palate
- implants: turn off the perio response to allow antibiotics to work and stop rejection
- stop inflam in perio disease
igg1 and iga, and boosters
igg1 is systemic antibody that floats in circulation. iga is secretory and coats the mucosal surface ONLY when you get APC presentation through mucosal immune system (GI or respiratory). stops transmission!
- can also be converted to ige that causes allergies!
booster:
- in vaccine, you get igg1. if infected, make iga
adjuvant in antibody production peanuts
oral delivery (peanuts) leads to immune suppression. NO ADJUVANT so no antibody production (harmful when IGG1-> IGE). this leads to immune suppression via treg. downregulates t and b cell.
- most common is aluminum
dna sequencing
sequencing: must create strands that stop at every nucleotide. separate via gel and see sequence. color coded and only one primer
- has no3’ OH so can’t add next nucleotide! therefore it keeps stopping
PCR comparison
uses 2 primers, annealing temp, uses taq polymerase,
needs copied dna, not fragments but can’t do quantative detection, only qPCR can
- this can’t tell you if virus is infectious or not! must lyse virus!
eliza comparison
dna is not quantitative and doesn’t cause disease, protein does!
- not amplified but very sensitive
- protein isn’t very stable
- no amplification so loose quantitation
- can only determine concentration so quantitative BUT can’t determine folded vs unfolded protein since only measures epitopes
- very sensitive. overcomes the limited quantity of sample by increasing signal
qpcr
count cycles of dna replication and run standard to compare the number of cycles, not counting the product but based off a standard curve
- quantitative!
- can measure viral load in saliva but not infected vs noninfected particles
restriction enzymes
endo cut within dna
- exo cut at restriction sites on the ends
- palindromic sites
- restriction fragment length polymorphism is that every person has different lengths of strands for restriction sites?
microbubble antigen detection tech
uses n antigen
- take swab, monoclonal capture antibody is coated onto any metal nanoparticle. sandwich forms with n antigen. wash beads with h202, form o2 bubbles, magnets, quantification
THIS tells you infection! good for knowing when to return from quarantine
- this is live virus
It is not sensitive enough to detect in sewer or urine samples. Instead, raman is used.
- bigger bubble means maturity of virus and more infectious
covid and ace 2
virus kills ace 2 enzyme, allowing it in, this is downreg of ace 2 then which leads to more angiotensin 2 which is acute lung and cardiac damage
- covid vax must cross lipid mem.
we WANT CVD protection (decrease hypertension and prolif, but instead covid gives CVD/hypertension by inncreasing angiotensin
low cost electrochemical advanced diagnostics
lead
- gold nanoparticles, uses spike protein!!
- coat graphite electrode with ace 2, add spike protein = charge changes current that can be measured.
- cheap but need lots of particles. not sensitive
- quantitative
enhanced raman scattering detection (SERS??)
- single virus detection
- no extraction or amplification = best
- cover the gold needles with ace 2, then respond to spike protein and shift angle, creating a raman shift that can be read
sensitive enough to detect in sewer or urine samples. - can detect in various public places
eliza and the covid problem
test shows decreased ace 2 activity, but higher ace 2 concentration in blood, leading to false covid cases@
- could be very off since only measure protein concentration
- HOWEVER if you use it to measure ang 1-7, then its much more accurate (product of ace 2 enzyme)
e coli protein production
can’t do post trans modification like humans
- they don’t have introns or splicing, diff reg sequences, no 5’ cap, poly a tail. coding sequence MUST be without introns
- instead have 5’ UTR and 3’ UTR
- solution is to copy mature transcripts and put them into good host cell, then change coding sequence for host dell. kill all bad cell that didn’t take up plasmid then grow and purify
yeast protein production
used for insulin but can’t make the c peptide
- results in neuropath, retinopathy
cho cells protein production
- can do post trans modification like humans@
plant and insect cell protein production
have a diff glycosylation profile
plasmid recombinance
- small size means restriction enzyme sites are more specific
- autonomous replication
- origin of replication determines the copy number
- multiple clonig site has dna sequencing primers that have synthetic restriction sites to insert stuff
- also has antibiotic resistant gene, which is how plasmids are selected
- must find mature transcription not the whole gene to insert into plasmid. remove introns using spliceosome
codon optimization for recombinant proteins
when you change the codon for prokaryotic system
3rd base wobbles so codon changes are possible determined using host cell tRNA so its not limited!
- each cell system has different proteases, cleave protein. if you know them, sites can be changed therefore change protein function, load ribosome etc
- goal of this is to make lots of protein by optimizing coding sequence, right host cell, copy #, plasmids etc.
endocrine disorders replacing deficient proteins
- insulin for dia 1
- growth hormone : somatotropin
- insulin like growth factor for bone regeneration
replacing def proteins in hemostasis and thrombosis
type a hemophilia are missing clotting factor 8
- type b lack 9
- protein c in venous thrombosis
- tissue plasminogen activator for embolism and stroke (TPA)
exenatide/ exendin
binds to glucagon like peptide in diabetes 2
- insulin resistance so decreased concentration. this protein helps to regulate the RELEASE of insulin into circulation and makes sure that the right amount is released
- can safely overdose on insulin, no hypoglycemia
EMDS
emdogain
- this is enamel matrix derivatives
- work in enamel, MMPS, amelogenins and ameloblastin
- tooth bud from piglet
- target: cementoblasts, osteoblasts, gingival epi cells, gingival fibroblasts, pdl cells
FGF-2
fibroblast growth factor 2. works in PDL migration
- target: cementoblasts, osteoblasts, gingival epi cells, gingival fibroblasts, pdl cells
PDGF- BB
PLATElet derived growth factor - BB
- effects gingival fibroblast and PDL fibroblast
- target: cementoblasts, osteoblasts, gingival fibroblasts, pdl cells
P- 15 ***
peptide -15
- small 15 aa peptide that mimics cell binding domain
- helps in osteoblast adhesion
- target: osteoblasts
BMP - 2
bone morphogentic protein
- PDL calcification
- target: mesenchymal stem cell, osteoblasts
IGF-1
insulin like growth factor
- osteogenic differentiation of human PDL stem cells
- bone regeneration in diabetic patients
- target: osteoblasts, pdl
GDF-5
growth differentiation factor 5
- target PDL cell (MMP) and mesenchymal stem cells
- osteogenesis
viral trap proteins
ace2 (spike protein) and fril (glycoprotein on envelope) trap oral viruses like covid, HSV, flu
enzymes for oral cavity pathogens
dextranase (breaks a 1-6), mutanase (a 1-3) to disrupt EPS
- lipase for candida albicans kills hyphae of fungi
inhibition of biofilm formation using topical PG1 BUT must penetrate EPS first using either enzyme or cationic
COMBO: best! but must have long contact time
cationic antimicrobial peptides that destroy bac cell membranes by creating dimple then lyse
fril
binds glycoproteins on most virus and aggregation to gum! even if virus enters, FRIL blocks by putting virus in endosome so less chance nucleus allows reproduction
- also can’t transmit. decrease viral load so neutralization in throat.
- more broad trapping can make gum
how to do oral drug delivery
- protein drugs are degraded in stomach
- gut epi is imperm to drugs (tight junctions)
- plants have b 1-4, 1-6 linkages that don’t digest so protected in stomach
- gut bacteria break down b bonds in gut lumen, releasing drug
- tags are needed to cross gut epi cells
- CTB - GM1 receptor binding (borrowed from other pathogens like cholera!!)
- protein transduction domain then bypasses receptors
oral drug delivery tags
CTB (cholera), PTD (HIV), DCpep (dendritic cells)
- gut is biggest absorption surface!
- tags deliver to spicific cells or tissues
covid ace 2 gum
Ace2 is regulatory enzyme in RAS pathway (cardioprotective). spike binds here.
CTB ace 2 binds ace2. CTB then can either bind directly to Ace2 and GM1 (competitive binding for covid) OR form microparticles and aggregate covid
other uses for combined ace2/ang 1-7 oral drug delivery
- dose dependent
- can stop AND REVERSE pulmonary hypertension
- preservation of normal right ventricular geometry (before its enlarged RV so decreased heart function)
adjuvants in vaccines
- adjuvant is present, steering immune response towards increasing antibody production
virus selection for gene therapy
first ID defective gene, crisper, then correct it using vector
small size is better, decreased restriction sites
- lower loading capacity is better
- long lasting is better than transient
- non integration better since can trigger oncogene but then it decreases duration
- best is adenoassociated virus and retrovirus
adenoassociated vectors
no viral coding sequence produced in absence of helper virus
- transduces nondividing cells in vivo (neurons?)
- long term expression in immunocomp animals
- need helper virus so it can’t just replicate itself!
structure: promoter, intron, therapeutic gene, polyA
polio and virus vector
increase in IgG igA
- increase in polio titers against all serotypes!
C1 therapy using AAV
- SILENCING
to attenuate alveolar bone - it is an atpase pump that is expressed
- mirna to suppress protein
- slow endodontic disease. regulates toll like receptor
d2 gene therapy
SILENCING to treat periapical disease
- decreases inflammation
- reduces bone resorption by reducing osteoclasts, enhancing bone formation by increasing inflam
- growth proteins
apoptosis
physiological death. done by capsases
- NO lysosome
- enzymes that degrade dna, important in biological processess like ageing embryogenesis, host defense,
- 2 pathways
- required for development
- xs leads to disease. especially in nonprolif cells
- insufficient apoptosis leads to xs cells
- 1 cell. doesn’t damage the whole tissue
necrosis
cell injury like lack of o2, atp, mechanical stuff, chem, lysosomal enzymes
- leads to membrane dissolution, xs ca uptake, phospholipase actication
- cell material released out = damage to other cells
- lysosome plays major role. inflammatory!
- cell swells and bursts
necroptosis
cell death involving TNF factors leading to receptor interacting protein kinases
- viral defense. caspase independent
pyroptosis
associated with inflammasome and caspase 1
- NOT cell death but maturation of cytokines
latrogenic
drugs that signal apoptosis
- cytotoxic, radiation, autoimmune disease, thyroid goiters
they interfere with cell cycle and mitosis
apoptosis pathway
- cells shrink cyto and organells condense
- chromatin condense and peripheral aggregation
- cell breaks up into apoptotic bodies (mem bound BLEB) with DNA and cyto organelles
- rapid removal by macrophages
*always membrane bound
caspases
- apoptosis
- used in mito and bcl-2
- cysteine proteases that cleave proteins after asp residues
- activated by cleavage (zymogen) or dimerization
extrinsic pathway caspase
- death receptor pathway
- procaspase 8 and 10 that work with 3 and 7
- TNF, Fas, and trail (DR4 is ligand) are ligands
- bind to adaptor proteins, then facilitate procapsid binding, dimer, = activated
initiator caspase
- 2,8,9,10 that are the first to be on. send signal
- made as monomers. conformatinoal change, dimerize and cleavage to activate effector caspases
effector caspases
3,6,7
- expressed as preformed dimers, activated by direct proteolytic cleavage
- cleave substrates, destroying cells
intrinsic caspase pathway
- mitochondrial/BCL-2
- no receptor or ligand.
- procaspase 9 then activates 3 and 7
- regulation of mito perm and release of death signals (cyto c)
- bcl 2 family promotes apoptosis, release effectors like bax and bad (balance determines death) so mito pores open up and leak out. release apoptotic factors, signal iniator caspase then executioner caspase 9
- regulators are bcl2, bclx that block effectors and apoptotic signal
apoptotic factors for intrinsic pathway
to stim it, you decrease atp, increase ROS, mem collapse
- then leaks out cyto c that forms apoptosome (indirect caspase actication) and AIF that is direct
- high mito swelling, perm transition pore complex is open (PTPC)
execution pathway of caspases
- then effector pathway is increases caspase 3,6,7, turn on endonuc and dna fragmentation happens
apoptosis features
- in PCR it forms ladder, necrosis is a smear
- formation of mem bound apoptotic bodies
0 happens when the phosphatidyl serine is switched from in to out!! resulting in annexin binding and phagosome formation
highly proliferative cells
bone marrow, epi, hepatocytes, testes, breast, fibroblasts
- cab be biopsied!
low turnover cells BUT can respond to stim (facultative dividers)
endothelial cells, supportive cells in organs, bone, cartilage, smooth muscle
- cab be biopsied!
cells with little or no capacity to divide
nervces, skeletal and cardiac muscle
how to cells know to proliferate
growth factors, mitogens (eco), hormones ( b and t cells), antigens, increased demand, injury
interphase, g0 and g1
g0: non prolif non dormant. just differentiated cells that can reenter cycle
g1: increase in mass, gives time to monitor internal and external environment for s phasge
can enter from g0 OR M
interphase, s and g2
s phase is chromosome duplication (46 with 92 chromatids, tetrapod). many origins of rep
- g2: additionally growth functions, monitor conditions for M phase (good chromosome duplication, preparation for mitosis)
control of enzymes and transcription factors in cell cycle
de novo protein syntehsis
protein modification like kinase, acetylation, changing location
protein degredation using ubiquitin (ER, done in proteosome)
cyclin dependent kinases
- serine threonine proten kinases
- regulation involves: cyclines, phospho, CDK inhibitors
- activity leads to downstream phospho of targets leading to changes in gene expression or protein function
- active in different phases, but their concentration remains the same
phosphorylation is done on threonine and tyrosine. can be active, inactive or change in location
cyclins
their levels rise and fall during cycle (synth and degredation) by ubiquitin.
- highly specific for each cdk
CDK inhibitors
regulated by internal and external signals
- INK4: inhibitor of CDK4 and 6. 4 of them (p15,16,18,19) block association with cyclin. G1!
- CIP/KIP family (P21, 27,57) inactivate ckd-cyclin complex. checkpoint regulator
g1 restriction point
checks state of cell and eco. requires size, eco (nutrients, growth factors). cell commits to cell cycle entry and chromosome duplication
g1, s and g2 checkpoint
detection of dna damage
- use common strategies to detect. regulate progression. to enter mitosis, need size and good dna
mitosis checkpoint
metaphase to anaphase transition
- checks if spindle attached, allows chromatid separation
failure to meet the restriction checkpoints
delay until right conditions OR exit from cell cycle and possible cell death
if repair fails, apoptosis, senescence or become malignant
- senescence means still function but no replication
- p21 cdk inhibition and GADD45 does dna repair
DNA damage response (DDR)
involves g1,g2, and 2
- sensors activate transducers that activate effectors leading to cell cycle blockage and dna repair
- ATM and ATR are sensors
- chk1 and 2 are transducer kinases and p53 (induces 21) a transcription factor
OR apoptosis OR malignant
external influences on cell cycle regulation
dna damage, cell signalling pathway (pi3k/akt is most common) and telomeres (promote formation of protein caps, limited # of cell cycles before cell gets senescent)
cancer cell cycle
- mutations lead. togenomic instability
- protooncogenes are normally growth. in cancer, become protoonco and gain of function
- tumor suppressors block cell cycle, loss of function lead to no reg and neoplasia
osteogenesis imperfecta, juvenile periodontitis, sickle cell anemia, covid
dna diagnosis diseases, rna for covid
infectious diseases, caries, cancer, covid
protein diagnosis
growth factors (FGF2, IGF1, BMP), osteogenesis, osteoblast diff, cementogenesis, angiogenesis, adhesion
targets for protein drugs in dental
human gene therapy SCID
retinal pigment epi protein def (lipase def)
- glybera injection to reduce lipoprotein
cancer immunotherapy
chimeric antigen receptor
- proteins designed to fuse antigen binding domains of antibodies to tcell molecules
- insert retroviral vectors and t cell transduction (detect cancer!)
ways to open dna
histone modification, chromatin remodelling, rna pol
covalent histone mods
L: AMP
A: M
S: P
A and P are open, M is closed
ROS, aging disease, encephalo myopath, lactic acidosis, stroke, myoclonic epilepsi, kearns sayre syndrome
mito diseases
types of polymerases and sense strands
1 is rRNA
2 is mRNA
3 is tRNA
*transferases, add nucleo to 3’ end
sense strand is complementary to template (antisense strand)
= essentially the sense strand is SAME but with U and not T
TBP initiation
recognizes dna tata, single subunit that folds into symmetrical structure,
DNA contacts: b strands are in minor groove, interaction with other components of TFIID occur at alpha helix, bends dna 80-100 degrees, separates DNA strands for RNA pol!
major and minor groove interactions
Major and minor groove have specific interactions with DNA proteins since don’t need to open the strands! H bonding, ionic and hydrophobic interactions
Major groove: h bonds with donors and acceptors (asparagine 51 is in major groove)
Ionic bonds and hydrophobic interactions are basic requisites for contact between dna and amino acids, transcription factors, make it stronger!
minor groove is b strands
major is a helix with basic aa
rna pol and pausing initiation transcription
largely need to bring in RNA pol. 1) bind regulators like transcription activators 2) recruit rna pol to promoter 3) release rna pol to begin transcription 4) release rna pol from pause (meaning it stops after transcribing xx nucleotides, common in humans)
repression of transcription
competetive dna binding (close together so cancel each other out), masking the activation surface, direct interaction with general transcription factors, recruitment of chromatin remodeling complexes (tighening chromosome), recruitment of histone deacetylases (close dna), recruitment of histone methyl transferase (close up dna)
helix-loop helix dimerization motifs
mediate dimerization and dna binding
stronger than bZip
- dimerize by hydrophobic interactions
- n terminal helix lies on major groove, basic aa stabilize
splicing pattern genes
denin sialoprotein and phosphoprotein made from a single DSPP
- also tropomyocin. cutting creates different muscle types
splicing pattern genes
denin sialoprotein and phosphoprotein made from a single DSPP
- also tropomyocin. cutting creates different muscle types
ERAD: cystic fibrosis, gauchers, pompes, hemophilia, diabetes
proten def disorder
trna and synthetase structure
lopps are single strands, acceptor stem is rigid since 2 strands
- mods include u pseudo uridine and d dihydro uridine
- binds to anticodon via h bond
- aa is loaded onto 3’ OH end by specific synthetase (only 20) using atp
synthetase has aa, trna and atp binding site
diptheria
blocks protein synth by binding ADP on eEF2, no translocation
protein recombinance production steps
- clone human genes w/o introns
- id reg sequence differences between prok and euk
- use plasmids for cloning
- optimize rare codons
- select cells expressing recombinant protein
- purification, formulation, injection
